Agreement of activity monitors for assessment of patients with sub-acute stroke in an inpatient rehabilitation facility.

PURPOSE: Determine the level of agreement of three activity monitors compared with the gold standard (video review) on the activity level of patients with stroke. METHODS: A prospective, observational, agreement study was performed on 47 individuals with sub-acute stroke in an inpatient rehabilitation facility. Data was collected during one physical therapy session. Individuals wore three device types; Actigraph (AG), Activpal (AP), and stepwatch activity monitor (SAM). Variables assessed were step counts for each limb (hemiparetic and non-hemiparetic) and percent time standing and other. ANALYSIS: Results from the activity monitors were compared to the video review and assessed for agreement using the intraclass correlation coefficient (ICC) and accuracy of mean difference from video observation. RESULTS: The step counts with the SAM on the non-hemiparetic limb had the highest ICC for step counts (ICC = 0.98, p < 0.001) and were overestimated with 21% accuracy. The SAM on the hemiparetic limb had 9.7% accuracy (ICC = 0.92, p < 0.001). For percent standing time all devices overestimated with poor reliability. For percent other activity time, the AP had the best accuracy and underestimated for both the hemiparetic limb (9.9% accuracy; ICC = 0.90, p < 0.001) and non-hemiparetic limb (8.3% accuracy; ICC = 0.84, p < 0.001). CONCLUSIONS: The use of multiple devices may be warranted to capture an accurate understanding of activity levels in this population of individuals with sub-acute stroke. There are concerns with all monitors and clinicians and researchers should be aware of what measures they are wanting to understand about their population.

The stepwatch activity monitor worn on the hemiparetic limb provided the best accuracy and excellent reliability for step counts in this population of subacute stroke.For percent standing time all devices overestimated with poor reliability.For percent other time, the AP had the best accuracy and good reliability on the non-hemiparetic limb.The use of multiple devices may be warranted to capture a more accurate understanding of activity level in this population of individuals with sub-acute stroke.Clinicians and researchers need to be aware of the biases of these devices in this population.

Indirect Excitons and Trions in MoSe2/WSe2 van der Waals Heterostructures.

Indirect excitons (IX) in semiconductor heterostructures are bosons, which can cool below the temperature of quantum degeneracy and can be effectively controlled by voltage and light. IX quantum Bose gases and IX devices were explored in GaAs heterostructures where an IX range of existence is limited to low temperatures due to low IX binding energies. IXs in van der Waals transition-metal dichalcogenide (TMD) heterostructures are characterized by large binding energies giving the opportunity for exploring excitonic quantum gases and for creating excitonic devices at high temperatures. TMD heterostructures also offer a new platform for studying single-exciton phenomena and few-particle complexes. In this work, we present studies of IXs in MoSe2/WSe2 heterostructures and report on two IX luminescence lines whose energy splitting and temperature dependence identify them as neutral and charged IXs. The experimentally found binding energy of the indirect charged excitons, that is, indirect trions, is close to the calculated binding energy of 28 meV for negative indirect trions in TMD heterostructures [Deilmann, T.; Thygesen, K. S. Nano Lett. 2018, 18, 1460]. We also report on the realization of IXs with a luminescence line width reaching 4 meV at low temperatures. An enhancement of IX luminescence intensity and the narrow line width are observed in localized spots.

The clinical significance of the POLG gene polymorphism in male infertility.

Based on association studies, an increasing number of gene polymorphisms have been proposed as modulators of spermatogenesis. Interestingly, a clear cause-effect relationship between a polymorphism of the POLG gene and oligo(astheno)zoospermia was recently described. The POLG gene contains a polymorphic CAG repeat, and the presence of a homozygous mutant (not10/not10 CAG) genotype was found only in infertile men. In the present study, a large number of infertile patients and normospermic men of Italian origin were studied to define the effect of POLG genotypes on spermatogenic potential and whether the homozygous mutant is specific for spermatogenic disturbances. The mutated genotype was found at the same frequency in both infertile and normospermic men. Mean values of sperm parameters such as sperm count, motility, and morphology did not differ significantly between carriers of the three different genotypes. Our study failed to confirm any influence of the POLG gene polymorphism on the efficiency of the spermatogenesis. More importantly, considering that the homozygous mutant genotype has been found in normospermic fertile men, the analysis of the CAG repeat tract of the POLG gene does not appear to have any clinical diagnostic value.

Genetic differentiation within and between populations of a hermaphroditic freshwater planarian.

Dispersal of individuals is an important factor that can influence genetic differentiation between populations. The hermaphroditic freshwater planarian Schmidtea polychroainhabits shallow regions of lakes and streams, in which they appear to be continuously distributed. In the present study we used three highly polymorphic markers for analysing small-scale and large-scale genetic structure within one, and between four natural lake populations. Genetic differentiation could already be observed between samples collected at least 13 m apart, but not between neighbouring samples, and was most pronounced between samples from different lakes. Probably due to the high variance in F(ST)values, a significant correlation between genetic differentiation and geographic distance could not be observed. These results show that individual dispersal of S. polychroa is limited, but that there is gene flow between subpopulations from the same lake. They further suggest that long-distance dispersal and gene flow between lakes, if present, is not a common process in S. polychroa.

In vitro analysis of nuclear transport mediated by the C-terminal shuttle domain of Tap.

The Tap protein of higher eukaryotes is implicated in the nuclear export of type D retroviral mRNA and some cellular mRNAs. Here we have developed an in vitro assay to study nuclear export mediated by the C-terminal shuttle domain of Tap involving the rapamycin-induced attachment of this transport domain to a nuclear green fluorescent protein-containing reporter. We found that export by the Tap transport domain does not involve cytosolic transport factors including the GTPase Ran. The transport domain directly binds to several nucleoporins positioned in different regions of the nuclear pore complex. These results argue that a direct interaction of the Tap transport domain with nucleoporins is responsible for its nucleocytoplasmic translocation. We found that the karyopherin beta-related export receptor CRM1 competes with the Tap transport domain for binding to Nup214 but not for binding to Nup62 or Nup153, suggesting that the Tap and CRM1 nuclear export pathways converge at the cytoplasmic periphery of the nuclear pore complex. Because the rates of in vitro nuclear import and export by the Tap transport domain are very similar, the directionality of mRNA export mediated by Tap probably is determined by mechanisms other than simple binding of the Tap transport domain to nucleoporins.

Nuclear pore complexes: dynamics in unexpected places.

In vivo studies on the dynamics of the nuclear pore complex (NPC) in yeast suggested that NPCs are highly mobile in the nuclear envelope. However, new evidence indicates that in mammalian cells NPCs are stably attached to a flexible lamina framework, but a peripheral component can exchange rapidly with an intranuclear pool.

Involvement of the lamin rod domain in heterotypic lamin interactions important for nuclear organization.

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle approximately 4/5 of its alpha-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import.

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.

Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1.

Receptor-mediated nucleocytoplasmic transport is dependent on the GTPase Ran and Ran-binding protein 1 (RanBP1). The acidic C terminus of Ran is required for high affinity interaction between Ran and RanBP1. We found that a novel Ran mutant with four of its five acidic C-terminal amino acids modified to alanine (RanC4A) has an approximately 20-fold reduced affinity for RanBP1. We investigated the effects of RanC4A on nuclear import and export in permeabilized HeLa cells. Although RanC4A promotes accumulation of the nuclear export receptor CRM1 at the cytoplasmic nucleoporin Nup214, it strongly stimulates nuclear export of GFP-NFAT. Since RanC4A exhibits an elevated affinity for CRM1 and other nuclear transport receptors, this suggests that formation of the export complex containing CRM1, Ran-GTP, and substrate is a rate-limiting step in export, not release from Nup214. Conversely, importin alpha/beta-dependent nuclear import of bovine serum albumin, coupled to a classical nuclear localization sequence is strongly inhibited by RanC4A. Inhibition can be reversed by additional importin alpha, which promotes the formation of an importin alpha/beta complex. These results provide physiological evidence that release of Ran-GTP from importin beta by RanBP1 and importin alpha is critical for the recycling of importin beta to a transport-competent state.

Nuclear import factors importin alpha and importin beta undergo mutually induced conformational changes upon association.

A heterodimer of importin alpha and importin beta accomplishes the nuclear import of proteins carrying classical nuclear localization signals (NLS). The interaction between the two import factors is mediated by the IBB domain of importin alpha and involves an extended recognition surface as shown by X-ray crystallography. Using a combination of biochemical and biophysical techniques we have investigated the formation of the importin beta:IBB domain complex in solution. Our data suggest that upon binding to the IBB domain, importin beta adopts a compact, proteolytically resistant conformation, while simultaneously the IBB domain folds into an alpha helix. We suggest a model to describe how these dual mutually induced conformational changes may orchestrate the nuclear import of NLS cargo in vivo.

Nup50, a nucleoplasmically oriented nucleoporin with a role in nuclear protein export.

We present here a detailed analysis of a rat polypeptide termed Nup50 (formerly NPAP60) that was previously found to be associated with the nuclear pore complex (F. Fan et al., Genomics 40:444-453, 1997). We have found that Nup50 (and/or a related 70-kDa polypeptide) is present in numerous rat cells and tissues. By immunofluorescence microscopy, Nup50 was found to be highly concentrated at the nuclear envelope of rat liver nuclei, whereas in cultured NRK cells it also is abundant in intranuclear regions. On the basis of immunogold electron microscopy of both rat liver nuclear envelopes and NRK cells, we determined that Nup50 is specifically localized in the nucleoplasmic fibrils of the pore complex. Microinjection of anti-Nup50 antibodies into the nucleus of NRK cells resulted in strong inhibition of nuclear export of a protein containing a leucine-rich nuclear export sequence, whereas nuclear import of a protein containing a classical nuclear localization sequence was unaffected. Correspondingly, CRM1, the export receptor for leucine-rich export sequences, directly bound to a fragment of Nup50 in vitro, whereas several other import and export receptors did not significantly interact with this fragment. Taken together, our data indicate that Nup50 has a direct role in nuclear protein export and probably serves as a binding site on the nuclear side of the pore complex for export receptor-cargo complexes.

Phosphorylation of the nuclear transport machinery down-regulates nuclear protein import in vitro.

We have examined whether signal-mediated nucleocytoplasmic transport can be regulated by phosphorylation of the nuclear transport machinery. Using digitonin-permeabilized cell assays to measure nuclear import and export, we found that the phosphatase inhibitors okadaic acid and microcystin inhibit transport mediated by the import receptors importin beta and transportin, but not by the export receptor CRM1. Several lines of evidence, including the finding that transport inhibition is partially reversed by the broad specificity protein kinase inhibitor staurosporine, indicate that transport inhibition is due to elevated phosphorylation of a component of the nuclear transport machinery. The kinases and phosphatases involved in this regulation are present in the permeabilized cells. A phosphorylation-sensitive component of the nuclear transport machinery also is present in permeabilized cells and is most likely a component of the nuclear pore complex. Substrate binding by the importin alpha.beta complex and the association of the complex with the nucleoporins Nup358/RanBP2 and Nup153 are not affected by phosphatase inhibitors, suggesting that transport inhibition by protein phosphorylation does not involve these steps. These results suggest that cells have mechanisms to negatively regulate entire nuclear transport pathways, thus providing a means to globally control cellular activity through effects on nucleocytoplasmic trafficking.

Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70.

Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process.

Transfusion-transmitted virus infection in HIV-1-seropositive patients.

OBJECTIVES: To investigate the prevalence, persistence and genome heterogeneity of transfusion-transmitted (TTV) in HIV-1-infected patients, a group at high risk both of contracting blood-borne viruses and having viral persistence relating to immunodepression. METHODS: Plasma samples from 238 HIV-1 seropositive subjects and 226 healthy blood donors were examined for TTV-DNA both by polymerase chain reaction (PCR) using primers from the conserved regions in the N22 clone and PCR using primers deduced from the untranslated region (UTR). Direct DNA sequencing and phylogenetic analysis were used to characterize 27 TTV isolates from HIV-1 patients or healthy controls. RESULTS: Using PCR with the UTR primers, TTV DNA was detected in a very high percentage (> 80%) of samples both from HIV-1 seropositive subjects and from blood donors. Using PCR with N22 primers, shown to detect viral strains associated with hepatitis of unknown etiology, TTV DNA was found in 103 of 238 (43.3%) HIV-1-infected patients and in 22 of 226 (9.7%) blood donors. There was no difference in the prevalence of the TTV DNA in HIV seropositive subjects with regard to clinical features related to immunosuppression, markers of HCV infection or intravenous drug use; presence of TTV DNA was associated significantly only with male gender (P = 0.003). Persistent or intermittent viremia was detected in plasma samples taken up over a period of 19 months in all (15 of 15) HIV-infected patients tested. CONCLUSIONS: The persistence and high frequency of infection detected by PCR with N22 primers in HIV-1 seropositive patients suggest that further clinical investigation of immunocompromised hosts will provide information to clarify the pathogenic role of TTV.

Human Nop5/Nop58 is a component common to the box C/D small nucleolar ribonucleoproteins.

We have identified an apparent human homolog of the yeast Nop5/Nop58 protein. hNop5/Nop58 codes for a protein of predicted molecular weight 59.6 kDa and is 46.8% identical to Saccharomyces cerevisiae Nop5/Nop58. Immunofluorescent staining with antibodies against hNop5/Nop58 indicate that it is localized primarily to the nucleolus, and coimmunoprecipitation from nuclear extracts demonstrates that hNop5/Nop58 interacts with the box C/D family of snoRNAs. Thus, hNop5/Nop58 is a common component of the box C/D snoRNPs, and joins fibrillarin as the second such component identified and characterized in metazoans.

A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export.

We recently developed an assay in which nuclear export of the shuttling transcription factor NFAT (nuclear factor of activated T cells) can be reconstituted in permeabilized cells with the GTPase Ran and the nuclear export receptor CRM1. We have now used this assay to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot hydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alone do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cytoplasmic periphery of the nuclear pore complex (NPC) in association with the p62 complex and Can/Nup214. RanGTP-dependent association of CRM1 with these nucleoporins was reconstituted in vitro. By biochemical fractionation and reconstitution, we showed that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC. Taken together, our results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that RanBP1 and probably RanBP2 are involved in the dissociation of nuclear export complexes from the NPC in a terminal step of transport.

All four homochiral enantiomers of a nuclear localization sequence derived from c-Myc serve as functional import signals.

The information that targets a protein to the nucleus often consists of a short cluster of basic amino acids called a nuclear localization sequence (NLS). Since a wide range of sequences rich in basic amino acid residues function as NLSs, we postulated that an NLS-like sequence composed exclusively of D-amino acids might have biological activity. We synthesized peptides corresponding to the c-Myc NLS composed of either all L or D-amino acids, both in the forward and reverse order. We tested these peptides for nuclear import activity in a digitonin-permeabilized cell assay. All four peptide-bovine serum albumin conjugates localized to the nucleus with similar efficiency, and each conjugate competed for import with an SV40 large T antigen-derived NLS conjugate. Cross-linking experiments with free NLS peptides in HeLa cytosol indicated that each peptide bound to a protein that migrated at the molecular weight of importin alpha. Recombinant importin alpha, importin beta, Ran, and NTF2 alone were sufficient to support the import of both L-form and D-form conjugates in permeabilized cells. This indicates that both D- and L-form NLS peptides use the same import machinery. Although the free D-forms of the NLS were proteolytically resistant in cytosol, the L-forms were rapidly degraded. To our knowledge, this is the first example of an intracellular pathway in which the receptor is insensitive to the chirality of the ligand.

cDNA cloning and analysis of the expression of nucleoporin p45.

The p62 complex is an assembly of four O-linked glycoproteins (p62, p58, p54, and p45) localized in the central region of the nuclear pore complex. It has been suggested to provide a substrate binding site near the central gated channel of the pore during nuclear protein import. The sequences of p62, p58, and p54 from rat have been reported previously. We have now carried out cDNA cloning of rat p45. The authenticity of the p45 clone was confirmed by two-dimensional gel analysis of the in vitro translated product of this clone. Sequence comparison showed that p45 is mostly identical to the amino terminal four-fifths of p58. p45 contains an N-terminal FG (Phe-Gly) repeat region, a middle coiled-coil region, and a truncated C-terminal FG repeat region (compared to p58). The sequence data and genomic Southern hybridization results strongly support the possibility that p45 and p58 are generated by mRNA alternative splicing. The sequences of three other p58-related cDNA clones indicate that the p58/p45 gene transcript gives rise to additional alternatively spliced mRNAs in mammalian cells. Interestingly, the expression level of p45 relative to p58 varies in different cultured cell lines, indicating that the p62 complex is heterogeneous with respect to these two subunits.